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Cloning and Characterization of Alcohol Dehydrogenase (adh) Promoter Region for Expression Under Submergence and Salinity Stress

Mohammad Arif Ashraf
Plant Biotechnology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka

Sudip Biswas
Plant Biotechnology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka

Samsad Razzaque
Plant Biotechnology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka

Taslima Haque
Plant Biotechnology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka

Zeba I Seraj
Plant Biotechnology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka

Abstract/ Executive Summary:

The characterization of promoter is important for developing stress tolerant crops as well as understanding the role of promoters in regulating gene expression. The current study was initiated with an aim to characterize the Adh promoter under salinity and submergence stress in rice calli. The upstream regions (~1kb) of the Adh gene was amplified from the genomic DNA of Arabidopsis (Columbia Ecotype). The amplified product was then cloned successively into an entry and promoter?characterization binary destination vector having the reporter gene β?glucuronidase (GUS) by applying Gateway Technology. A positive clone was confirmed by applying PCR, restriction digestion and sequencing. The construct was then transformed into Agrobacterium tumefaciens LBA4404 strain and rice calli infected with the latter. In both salt and submergence stresses, Adh could selectively express GUS gene activity up to two?fold compared to control.

Keywords: Stress inducible promoter, Alcohol dehydrogenase (Adh), Cloning

Location:

Start Date:

End Date:

Program Area: Risk Management in Agriculture

Non-commodity: Adoption of technology

Objectives:

In the current study, Adh promoter was cloned from Arabidopsis thaliana (Columbia ecotype) in an attempt to characterize its efficiency in salinity and submergence stress response. Calli generated from tissue culture responsive rice landrace, Binnatoa, was transformed with the cloned Adh promoter and GUS assay was conducted to measure the expression of Adh during submergence and salt stress.

Methodology:

Arabidopsis thaliana seeds were germinated to grow up to maturity. DNA was isolated using the CTAB method (Stewart and Via 1993). The extracted DNA was quantified using Nanodrop® spectrophotometer ND?1000 (Thermo Fisher Scientific Inc. Waltham, MA, USA). Target size (1009 bp) from Adh promoter was amplified by PCR with target specific primers. PCR reaction program for amplifying ~1kb Adh promoter was optimized as follows: Initial denaturation was at 95°C for 5 min and 35 cycles of denaturation at 95°C for 1 min, annealing at 61.5°C for 1 min, extension at 72°C for 2.10 min followed by a final extension at 72°C for 10 min. A final concentration of 2.3 mM MgCl₂, 0.1 mM dNTPS, 0.3 μM of each primer and 1 unit of recombinant Taq polymerase (Invitrogen, Carlsbad, CA, USA) was used. The forward primer was designed adding CACC overhang to ensure compatibility with pENTR/D?TOPO vector. PCR amplicons were gel extracted using Qiaquick Gel extraction kit (Qiagen, Hilden, Germany) and quantified through nanodrop. Cloning reaction into pENTR/D?TOPO vector (Invitrogen, Carlsbad, CA, USA) was then initiated following manufacturer’s protocol. The pENTR_Adh plasmid construct was transformed into E. coli DH5α competent cells through heat shock employing standard protocols (Sambrook et al. 1989). Successful cloning into pENTR was then confirmed by PCR, restriction digestion with NdeI restriction enzyme (NEBr inc.,Ipswich, MA,USA) followed by final confirmation by direct sequencing of the insert with gene specific primers. Cloned pENTR vector was then used to recombine the desired sequence of Adh promoter into the destination vector (pHGWFS7.0) using the Gateway® LR recombination reaction (Invitrogen, Carlsbad, CA, USA). The LR reaction was carried out following the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). The Gateway technology allows taking advantage of the site specific recombination properties of bacteriophage lambda to provide a rapid and highly efficient way to recombine target sequences to multiple vector systems (Invitrogen, Carlsbad, CA, USA). The destination vector (pHGWFS7.0) used here is efficient for promoter expression analysis (Karimi et al. 2002). Site specific recombination properties of Gateway system allowed recombination of the Adh promoter from the pENTR_Adh to the target destination vector (pHGWFS7.0). The successful recombination would place the Adh promoter immediately upstream of the GUS gene. Positive colonies were determined by PCR with gene specific primers and restriction digestions with NdeI (NEBr inc., Ipswich, MA, USA). Finally, Agrobacterium tumefaciens (LBA4404) was electroporated with the constructed pHGWFS7.0_Adh using standard protocols (Sambrook et al. 1989). Positive colonies were authenticated by PCR reactions with target?specific primers

Source of Information: Plant Tissue Cult. & Biotech. 24(1): 111-120, 2014 (June)

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Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Binnatoa calli were used for the transformation with Agrobacterium containing the pHGWF7.0_Adh binary vector. Tissue culture responsive Binnatoa calli could easily be transfected and GUS expression was examined to observe the activity of the Adh promoter. Control wild type explant gave no color and calli with Adh promoter without stress showed poor expression. The stressed explants showed up to two fold better expression compared to control and nostress explants. In salinity stress screening, GUS activity was two?fold higher in 10 hrs stress and in 5 hrs stress the expression was one and a half?fold higher compared to the control explants. In addition, GUS activity in submergence screening was found similar to salinity stress. The expression of GUS after 10 hrs was also two?fold higher and 5 hrs stress induced GUS expression was one and a half?fold higher compared to control. The study conducted here shows the inducibility of the Adh promoter under abiotic stresses like salinity and submergence. These results can be further fine tuned using quantitative measures of GUS activity as well as checking induction in the specific tissues of plants regenerated from the positive calli

Knowledge Management: Journal